Cardiac excitability is determined by the activity of several membrane currents including IKr, which is responsible for the terminal repolarization of the ventricular action potential. When IKr is disrupted by mutations in the HERG gene or blocked by drugs, life-threatening arrhythmias can result. Expression of HERG in frog oocytes produces currents with the biophysical and pharmacological properties of IKr, suggesting that HERG subunits are key components of the native channel. HERG channels exhibit a rapid inactivation that suppresses the current during depolarization; during repolarization the channels recover from inactivation and pass through the open state prior to closing. Because deactivation is slow, HERG current reaches its maximum during repolarization of the cardiac action potential and helps to ensure that repolarization is complete. The long-range goals of this work are to understand the gating mechanisms that specialize HERG for its physiological role in the heart. The N terminus has recently been found to regulate deactivation rate, and naturally-occuring alternative N termini lead to functional diversity among channels encoded by HERG and Merg1, the HERG ortholog in mouse. The modulation of deactivation by the N terminus arises from a domain that is spatially separable from a second domain that promotes C-type inactivation. Both of these functions are disrupted by modifications of the S4-S5 loop, which phenocopies a deletion of the N terminus. The aims of this proposal are to characterize the mechanism by which the N terminus regulates deactivation, to identify modifiers of gating using the yeast two-hybrid system and to characterize the electrophysiological properties and regional distribution within the heart of channels encoded by HERG2 and HERG3, two newly-identified genes within the HERG subfamily. If the aims of this proposal are achieved a more detailed picture of HERG gating will emerge along with a greater understanding of the physiology of HERG-related channels in the heart.